1. Field of the Invention
The present invention relates to a method for sustained expression of an exogenous gene, and more particularly in sustained expression of an exogenous gene by using baculovirus.
2. Description of the Prior Art
Gene delivery vectors for mammalian cells are classified into non-viral vectors and viral vectors. Non-viral methods of introducing an exogenous gene include various chemical agents and physical methods, e.g. polymer or liposome-mediated transfection or electroporation. However, these methods have following disadvantages including (1) low transfection efficiency for various cell types, especially the primary cells; (2) requirement for a large amount of purified exogenous gene; (3) damage to the cell membranes and disturbance of the cellular physiology.
Viruses possess a number of advantages as an exogenous gene delivery vector, that own high infection efficiency in a wide variety of cell types and tissues, and are provided with an ability of target specificity and enhanced expression of the exogenous gene in vitro and in vivo. Current viral vectors including retroviruses, lentivirus, adenoviruses, adeno-associated viruses and herpesvirus have been employed for gene therapy.
Although the above-mentioned vectors had certain degrees of success in particular application, there are still a variety of obstacles that cannot be surmounted. Plasmid delivery is considered less efficient than viral vectors. Retroviruses integrate viral genes into host cell chromosome resulting in the insertional mutagenesis in host cells or other dysfunction. The French team (2002) found that 9 X linked-SCID patients are cured by using retroviruses as gene delivery vector, but 2 patients were found with leukemia; therefore, there are some safety concerns in adopting retroviruses as gene delivery vectors.
As for adenoviruses, adenoviruses do not integrate viral genes into the host cells but may have safety issues concerning the elicitation of immune responses; in addition, there was a death report in a gene therapy experiment employing adenovirus (1999). Recently, a gutless vector has been developed for mitigating immune responses; however, it needs a helper virus for co-infection in the process of gutless vector production, resulting in more complexity of purifying viral particles. Though adeno-associcated viruses (AAV) is safer and enables longer gene expression time, adeno-associcated viruses can not deliver large genes because of their own 3.5-4 kb cloning capacity limit, and AAV production can not easily be scaled up due to complicated preparation procedure. Furthermore, adeno-associated viruses are found to integrate viral genes into the host cell chromosome and induce hepatoma in neonatal mice at high viral dosage. Due to these drawbacks, the lack of an ideal vector has been impeding successful application of gene therapy.
On the other hand, baculovirus (Autographa californica multiple nucleopolyhedrovirus) is a DNA virus that infects insects as the natural hosts, and has been widely employed for recombinant protein production. However, baculovirus is found to transduce various mammalian cells efficiently, e.g. BHK, CHO, CV-1, HeLa, 293, Cos7, neural cells, human fibroblasts and pancreatic cells, and can drive the expression of exogenous gene under promoters that are active in mammalian cells, e.g. SV-40 (Simian Virus 40) or CMV-IE (cytomegalovirus immediate-early) promoter, without any visible cytopathic effect (CPE). (1995). Besides, it is recently found that baculovirus can transduce bone marrow mesenchymal stem cells with transduction efficiency up to 87%.
Baculovirus vector has following advantages: (1) baculovirus does not replicate and induce cellular toxicity after transducing mammalian cells. Its DNA does not integrate into the host chromosome and is degraded with time; (2) there are no pre-existing antibodies against baculovirus in mammals since the natural hosts of baculovirus are insect cells; (3) baculovirus genome (˜130 kb) is much larger than other viral vectors and therefore is able to carry larger and more exogenous genes (at least 38 kb) into cells; (4) baculovirus production can be scaled up by infecting the natural hosts (insect cells) in simple operating procedures; and (5) baculovirus are classified into insect virus and can be operated in bio-safety 1 laboratories. Features (1) to (3) make baculovirus an effective and safer gene delivery vector, features (4) and (5) make baculovirus scaled up in a safe and easy way. These features altogether make baculovirus a good gene therapy vector in mammalian cells. Thanks to these features, baculovirus has hence been developed as a candidate for exogenous gene vector, but a number of roadblocks need to be lifted. One major hurdle is transient expression nature (<7 days) due to its inability to replicate in mammalian cells and has a large genome with multiple recognition sites for many restriction endonucleases. Since the genome of baculovirus cannot replicate in mammalian cells, it is usually degraded or diluted soon after transduction, leading to loss of transgene expression over time.
In general, there are two ways for sustained expression and replication of an exogenous gene in cells. First, the exogenous gene is integrated into host chromosome for the exogenous gene to be replicated with the cellular chromosome. As mentioned above, this method might easily interfere with the normal gene function of cells.
The other method for sustained expression and replication of an exogenous gene is to form a replicable episomal plasmid, wherein the episomal plasmid carries the exogenous gene and includes an origin of replication sequence that is able to allow the episomal plasmid to self-replicate and stably express the exogenous gene in mammalian cells. The episomal plasmid prevents dysfunctional cell metabolism without integrating into the cellular chromosome.
To sum up, the primary goal of this invention to develop a baculovirus vector that allows for the formation of a replicable episomal plasmid for sustained expression of exogenous genes in mammalian cells.